The study of oligonucleotides has become a key area of interest for many reasons including potential uses in therapeutic and diagnostic applications (Agrawal, S., TIBTECH, 1996, 14, 375-382; Marr, J., Drug Discovery Today, 1996, 1, 94-102; Rush, W., Science, 1997, 276, 1192-1193). One of the more interesting applications of oligonucleotides is the ability to modulate gene and protein function in a sequence specific manner. A direct result of studying oligonucleotides including their analogs in variety of applications is the need for large quantities of compounds having high purity. Presently, the synthesis of oligonucleotides and their analogs remains a tedious and costly process. There remains an ongoing need in this area for developing improved synthetic processes that facilitate the synthesis of oligonucleotides.
Phosphoramidites are important building blocks for the synthesis of oligonucleotides. The most commonly used process in oligonucleotide synthesis using solid phase chemistries is the phosphoramidite approach. In a similar process the support used is a soluble support (Bonora et al., Nucleic Acids Res., 1993, 21, 1213-1217). The phosphoramidite approach is also widely used in solution phase chemistries for oligonucleotide synthesis. Deoxyribonucleoside phosphoramidite derivatives (Becaucage et al., Tetrahedron Lett., 1981, 22, 1859-1862) have also been used in the synthesis of oligonucleotides.
Phosphoramidites for a variety of nucleosides are commercially available through a myriad of vendors. 3'-O-phosphoramidites are the most widely used amidites but the synthesis of oligonucleotides can involve the use of 5'-O- and 2'-O- phosphoramidites (Wagner et al., Nucleosides & Nucleotides, 1997, 17, 1657-1660; Bhan et al., Nucleosides & Nucleotides, 1997, 17, 1195-1199). There are also many phosphoramidites available that are not nucleosides (Cruachem Inc., Dulles, Va.; Clontech, Palo Alto, Calif.).
One of the steps in the phosphoramidite approach to oligonucleotide synthesis is the 3'-O-phosphitylation of 5'-O-protected nucleosides. Additionally, exocyclic amino groups and other functional groups present on nucleobase moieties are normally protected prior to phosphitylation. Traditionally phosphitylation of nucleosides is performed by treatment of the protected nucleosides with a phosphitylating reagent such as chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine which is very reactive and does not require an activator or 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (bis amidite reagent) which requires an activator. After preparation the nucleoside 3'-O-phosphoramidite is coupled to a 5'-OH group of a nucleoside, nucleotide, oligonucleoside or oligonucleotide.
The activator most commonly used in phosphitylation reactions is 1H-tetrazole. There are inherent problems with the use of 1H-tetrazole, especially when performing larger scale syntheses. For example, 1H-tetrazole is known to be explosive. According to the material safety data sheet (MSDS) 1H-tetrazole (1H-tetrazole, 98%) can be harmful if inhaled, ingested or absorbed through the skin. The MSDS also states that 1H-tetrazole can explode if heated above its melting temperature of 155.degree. C. and may form very sensitive explosive metallic compounds. In addition, 1H-tetrazole is known to Hence 1H-tetrazole requires special handling during its storage, use, and disposal.
Aside from its toxicity and explosive nature 1H-tetrazole is acidic and can cause deblocking of the 5'-O-protecting group and can also cause depurination during the phosphitylation step of amidite synthesis (Krotz et al., Tetrahedron Lett., 1997, 38, 3875-3878). Inadvertent deblocking of the 5'-O- protecting group is also a problem when chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine is used. Recently, trimethylchlorosilane has been used as an activator in the phosphitylation of 5'-O-DMT nucleosides with bis amidite reagent but this reagent is usually contaminated with HCl which leads to deprotection and formation of undesired products (Dabkowski, W., et al. Chem. Comm., 1997, 877). The results for this phosphitylation are comparable to those for 1H-tetrazole.
Activators with a higher pKa (i.e., less acidic) than 1H-tetrazole (pKa 4.9) such as 4,5-dicyanoimidazole (pKa 5.2) have been used in the phosphitylation of 5'-O-DMT thymidine (Vargeese, C., Nucleic Acids Res., 1998, 26, 1046-1050).
A variety of activators have been used in the coupling of phosphoramidites in addition to 1H-tetrazole. 5-Ethylthio-1H-tetrazole (Wincott, F., et al., Nucleic Acids Res. 1995, 23, 2677) and 5-(4-nitrophenyl)-1H-tetrazole (Pon, R. T., Tetrahedron Lett., 1987, 28, 3643) have been used for the coupling of sterically crowded ribonucleoside monomers e.g. for RNA-synthesis. The pKa's for theses activators are 4.28 and 3.7 (1:1 ethanol:water), respectively. The use of pyridine hydrochloride/imidazole (pKa 5.23 (water)) as an activator for coupling of monomers was demonstrated by the synthesis of a dimer (Gryaznov, S. M., Letsinger, L. M., Nucleic Acids Res., 1992, 20, 1879). Benzimidazolium triflate (pKa 4.5 (1:1 ethanol:water)) (Hayakawa et al., J. Org. Chem., 1996, 61, 7996-7997) has been used as an activator for the synthesis of oligonucleotides having bulky or sterically crowded phosphorus protecting groups such as aryloxy groups. The use of imidazolium triflate (pKa 6.9 (water)) was demonstrated for the synthesis of a dimer in solution (Hayakawa, Y.; Kataoka, M., Nucleic Acids and Related Macromolecules: Synthesis, Structure, Function and Applications, Sep. 4-9, 1997, Ulm, Germany). The use of 4,5-dicyanoimidazole as an activator for the synthesis of nucleoside phosphoramidite and several 2'-modified oligonucleotides including phosphorothioates has also been reported (Vargeese, supra.).
Another disadvantage to using 1H-tetrazole is the cost of the reagent. The 1997 Aldrich Chemical Company catalog lists 1H-tetrazole at over ten dollars a gram for 98% material. The 99+% pure material lists for over forty seven dollars per gram. This reagent is used in excess of the stoichiometric amount of nucleoside present in the reaction mixture resulting in considerable cost especially during large scale syntheses.
The solubility of 1H-tetrazole is also a factor in the large scale synthesis of phosphoramidites, oligonucleotides and their analogs. The solubility of 1H-tetrazole is about 0.5 M in acetonitrile. This low solubility is a limiting factor on the volume of solvent that is necessary to run a phosphitylation reaction. An activator having higher solubility would be preferred to allow the use of minimum volumes of reactions thereby also lowering the cost and the production of waste effluents. Furthermore, commonly used 1H-tetrazole (0.45 M solution) for oligonucleotide synthesis precipitates 1H-tetrazole when the room-temperature drops below 20.degree. C. Thus, blocking the lines on the automated synthesizer.
Due to ongoing clinical demand (See, for example, Crooke et al., Biotechnology and Genetic Engineering Reviews, 1998, 15, 121-157) the synthesis of oligonucleotides and their analogs is being performed utilizing increasingly larger scale reactions than in the past. One of the most common processes used in the synthesis of these compounds utilizes phosphoramidites that are routinely prepared and used in conjunction with an activator. There exists a need for phosphitylation activators that poses less hazards, are less acidic, and less expensive than activating agents that are currently being used, such as 1H-tetrazole. This invention is directed to this, as well as other, important ends.